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. 2005 Jul;79(13):8361-73.
doi: 10.1128/JVI.79.13.8361-8373.2005.

Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion

Marco Patrone  1 , Massimiliano Secchi, Loretta Fiorina, Mariagrazia Ierardi, Gabriele Milanesi, Andrea Gallina
Affiliations

Human cytomegalovirus UL130 protein promotes endothelial cell infection through a producer cell modification of the virion

Marco Patrone et al. J Virol. 2005 Jul.

Abstract

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.

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Figures

FIG. 1.
FIG. 1.
(A) Scheme of the UL131A-128 locus in the reference endotheliotropic strain VR1814 and the vaccine strain Towne. UL131A is interrupted by one intron. UL130 is contained in the central exon, surrounded by the UL131A distal coding tract and the UL128 first coding tract. UL128 is interrupted by two introns. Towne bears a 2-bp insertion within the UL130 distal tract, which creates a −1 frameshift replacing the 3′-most 12 codons with a 26-codon extension. Colors and symbols: striped boxes, UL130 N-terminal signal sequence coding regions; gray boxes, tracts devoid of similarity to known genes; black boxes, putative chemokine-like domain coding regions; checkerboard box, Towne UL130 missense extension; aataaa, polyadenylation signal; fs, frameshift mutation. (B) VR1814 UL130 nucleotide sequence and encoded amino acids (16). Lowercase, bold letters, start/stop codons; underlined amino acids, signal sequence; uppercase, bold letters, chemokine-like domain (29); C, putative chemokine cysteines in pUL130; NXT, N-linked glycosylation sites. The 3′ end of Towne UL130 is also shown in order to highlight the 2-nucleotide insertion (boxed) causing the reading frameshift and expansion to the UL128 start codon. The nucleotide numbering in panel B corresponds to that of the FIX BAC clone derived from the VR1814 genome (accession number AC146907 [27]) and to that of a partial Towne sequence (accession number AY446869 [7]).
FIG. 2.
FIG. 2.
The UL130 protein is translocated into the ER lumen and N-glycosylated, and its signal sequence is shortened in vitro. The UL130 (VR1814) gene or a deletion mutant lacking amino acids 2 to 25 (Δ2-25) was subjected to coupled in vitro transcription-translation in rabbit reticulocyte lysates, supplemented or not with canine pancreatic microsomes for cotranslational translocation-glycosylation. Abbreviations: endo F, N-glycosidase F treatment; cfg, centrifugation of the translation mixture through a buffered sucrose cushion at 10,000 × g; P10, centrifugation pellet; S10, centrifugation supernatant. The cartoon colors and symbols are the same as those described in the legend to Fig. 1. *, N-linked glycosylation sites.
FIG. 3.
FIG. 3.
Western blot (WB) analysis of human cells expressing UL130. (A) pUL130 glycosylation state in UL130-transfected HeLa cells. nt, not treated; tun, overnight treatment with 5 μg/ml tunicamycin. (B) pUL130 glycosylation mutants in HeLa cells. wt, wild-type UL130 product. (C and D) pUL130 glycosylation in HCMV-infected HELF (5 dpi) (C) and HUVEC (D) lysates; in panel D, the lane profile plots and percentages of areas under the peaks refer to the right-most lane. For untreated samples in panels A (left), B, and D, the underglycosylated forms of pUL130 are not observed.
FIG. 4.
FIG. 4.
The Towne UL130 protein is inherently unstable. (A, top) The lanes under the “HELF” label correspond to HCMV-infected (5 dpi) HELF lysates, whereas “HeLa” indicates UL130-transfected HeLa lysates. mi, mock infected. (Bottom) Reprobing with antibodies against the HCMV tegument protein pp65. (B) HeLa transfectants accumulate low levels of the Towne mutant UL130 protein (pUL130fs) relative to VR1814 pUL130 and the equivalent protein of the endotheliotropic Towne revertant (Towne rev), in which the frameshift was corrected by an exact back mutation (12, 16). Two replicate lanes each are shown for Towne rev and Towne. The Towne rev lanes were underloaded (1/10 the total protein used in the adjacent Towne and VR1814 lanes) for a better appreciation of the pUL130fs upshift. (C, D, and G) pUL130fs decays rapidly in infected HELF (C), transfected HeLa cells (D), and nucleofected HUVEC (G). Protein synthesis in the experiments shown in panels C, D, and G was arrested with 50 μg/ml cycloheximide (CHX) for increasing times prior to WB analysis. (E) pUL130fs is stabilized in HeLa cells by the lysosomal protease inhibitors pepstatin and leupeptin (lysos inh), but not by the proteasome inhibitor lactacystin, which effectively protects cyclin B1 (bottom) in the same cells.
FIG. 5.
FIG. 5.
UL130 transcomplementation in HELF. Virus strains were grown in mock-complementing HELF (VR1814 and Towne) or UL130-complementing HELF (Towne 130) for 10 days, starting from an MOI of 0.1, in replicate experiments. (A) The infectivities of both crude and purified virus preparations for HUVEC and HELF were determined in parallel by assaying IE-positive cells at 24 hpi. Abbreviations: cell, cell-free virus from cell sonication; rel, released virus; pur, purified virus; cen, centrifuged medium; rec (reconstituted), purified virions reassociated with the corresponding medium. The data are shown as HUVEC/HELF relative infectivities (mean HUVEC IU ml−1 + 95% confidence interval/mean HELF IU ml−1; n = 3). (B) Microscope fields of HUVEC infected with a multiplicity of 5 HELF IU/HUVEC and then immunohistochemically stained for IE1-pp72 at 24 hpi. The Towne images (middle panels) were purposely chosen to include positive nuclei to attest to the virus addition; a random field would have been empty, on average. Magnification, ×25.
FIG. 6.
FIG. 6.
Single-cycle viral output by VR1814, Towne, and Towne produced in the presence of pUL130, which were used to infect HUVEC. HUVEC monolayers were infected with each virus at 10 HELF IU/cell as described in Materials and Methods. After exposure of the cells to the viruses, pooled human sera were added to limit the infection to a single cycle of replication. After 2 days, the medium was changed to remove the blocking antibody. At 3 dpi, the medium was collected and HUVEC monolayers were stained for an IE antigen. The titers of HELF IU in the collected media were determined in parallel and plotted as HELF IU per infected HUVEC (A). Alternatively, the blocking antibody was left throughout the experiment and the virion tegument pp65 antigen released into the medium was measured by the quantification of WB lanes (B). The data are expressed as means ± 95% confidence intervals (n = 5).
FIG. 7.
FIG. 7.
UL130 transcomplementation in HUVEC. HUVEC harboring either retrovirally transduced UL130 (HUVEC 130) or a void retroviral vector (HUVEC mock) were infected with VR1814, Towne, or Towne 130. (A) Cells were infected with 5 HELF IU/HUVEC, fixed at 24 hpi, and stained for IE1-pp72. (B) Cells were infected with 0.5 HELF IU/HUVEC, fixed at 168 hpi, and stained as described above. Magnification, ×10.
FIG. 8.
FIG. 8.
The UL130 protein is found in virions. (A) The UL130 protein cofractionates with MCP in VR1814 particles sedimented through a Nycodenz gradient. Fraction 1 is the lightest fraction. (B to D) Particles from pooled fractions 11 to 13 were exposed to the indicated treatments. For panel C, as a control of endo H's effectiveness, HeLa lysates containing pUL130 were treated alone or after being mixed with VR1814 particles (lanes HeLa and VR1814+HeLa, respectively). In panel D, pUL130 exhibits a quantitative, though small, mobility shift after trypsin treatment. This suggests that it was cut at a basic residue close to either extremity; lysine-43 was the most probable cleavage site, as arginine-196 is upstream of the most C-terminal glycosylation site, whose removal should cause a larger mobility change. (E) Towne virions from a gradient similar to that used for panel A were analyzed for pUL130 alongside equal amounts of VR1814 virions and HeLa UL130 transfectants as controls. Abbreviations: nt, not treated; TX-100+ucfg, incubation with 0.5% Triton X-100, followed by ultracentrifugation and pellet analysis.
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